A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome has been constructed by insertion of the SV40 regulatory region (nt 5171-5243-270) into a deletion in the AAV genome from nt 144-264. The deletion removes the leftward most AAV promoter and about 100 bases upstream, but leaves intact in the hybrid genome the cap site of the transcript from the deleted AAV promoter. The inserted sequence contains the SV40 origin of replication (ori). However, when transfected into cells that constitutively express the SV40 T-antigen the plasmid replicates very poorly. We have discovered the inhibition of replication requires a trans-acting product from the AAV rep gene (an open reading frame in the left half of the genome whose products are necessary for replication) and two cis-acting target sequences which are within the inverted terminal repeats of the AAV genome. This proposal describes experiments in cell culture and in vitro to characterize the mechanism of this negative regulation. In cell culture experiments we will determine: 1) the exact parameters of the target sequence, 2) whether the distance between SV40 ori and the target sequence is important, 3) whether there is a critical ratio of oris to target sequences, 4) whether inhibition can be overcome by excess T-antigen, 5) which part of the AAV rep gene encodes the inhibitory product and 6) whether inhibition can be overcome by viral oncogene expression, or treatment of cells with physical or chemical carcinogens. We plan to use the established in vitro assay for SV40 DNA replication as an assay during fractionation of the inhibitory activity from infected cells. Experiments are described to isolate that AAV rep gene products from either infected cells or by means of expression vectors. There will also be an attempt to synthesize active rep gene products in vitro. By these means we hope to gain insight into the mechanisms underlying the negative regulation of AAV DNA replication.